Our work focuses on specialized synapses and circuitry in the inner retina. This year represents a major transition in the lab, and we are working hard to put various types of technology in place that will drive our experimental approach for years to come. We have expanded our study of inhibitory synaptic connections made by amacrine cells within the inner retina, to understand how feedforward and feedback inhibition contributes to signal processing in this network. We previously discovered that A17 amacrine cells provide rapid GABAergic feedback to rod bipolar cell ribbon synapses via a release process that is independent of membrane depolarization or voltage-gated calcium channels (Chavez, et al., 2006). This rapid feedback may be essential to prevent the rapid depletion of readily-releasable vesicles from the rod bipolar cell synaptic terminal (Singer and Diamond, 2006). Our recent work indicates that this feedback extends the range over which these synapses encode luminance and compute contrast (manuscript in preparation). In addition, another study indicates that feedback inhibition enhances the gain of synaptic responses in the rod pathway to the absorption of single photons (manuscript submitted). In addition, we have combined electrophysiological and anatomical (EM) data with mathematical simulations to explore the role of the ribbon in regulating the delivery and release of vesicles at the presynaptic membrane. Our findings suggest that the ribbon may achieve a conveyor belt like function simply with passive diffusion of vesicles along the two-dimensional ribbon. These simulations also make testable predictions regarding the molecular mechanism by which the vesicles diffuse along the ribbon. A manuscript is fully developed and is nearing submission. We are combining electrophysiology, imaging approaches and cellular/network modeling to explore dendritic integration in directionally-selective ganglion cells. We are also taking advantage of numerous transgenic mouse lines to image specific cell types, which will enable us to record from synaptically coupled cone bipolar - ganglion cell pairs. This has enabled us to examine how directional selectivity is encoded at various stages of the circuit. This work also suggests that NMDA receptors play a unique role in ganglion cell dendrites to amplify the directionally selective signal. A manuscript is in preparation. We are also working to understand how NMDA receptors contribute to the developmental refinement of ganglion cell dendrites, with are precisely stratified within the inner plexiform layer, the synaptic neuropil of the inner retina. We have successfully combined multiple genetic tools to acquire a mouse in which the NMDA receptors can be knocked out in a single type of ganglion cell at any time during development. This project has taken a long time to prepare, but initial experiments with the mice are very promising and suggest that we will gain exciting new insights in the role of NMDA receptors in retinal development. Our work on feedback inhibition has led us to undertake a long-term effort to understand, in a systematic way, how amacrine cells contribute to visual signaling in the inner retina. With over 40 different kinds of amacrine cell, this prospect can be a bit overwhelming. To start, we have identified narrow- and wide-field amacrine cells that can be identified and manipulated by genetic means. We have acquired mouse lines in which CRE is expressed specifically in certain amacrine cells and then cross them with floxed lines enabling the CRE-expressing neurons to be silenced chemically (through CRE-dependent expression of designer receptors exclusively activated by designer drugs). The impact of this silencing on ganglion cell signaling will be examined with a microelectrode array. In addition, the cell-specific expression of calcium and voltage indicators will enable us to examine dendritic signaling in these cells that likely underlies visual processing in these cells (see Grimes, et al., 2010) that is not accessible with somatic electrophysiological recordings. We are also studying how the biophysical properties of synapses and neurons change with different phases of the circadian cycle or dark adaptation. Initially we have focused on changes in BK channel function in AII amacrine cells, which play distinct roles in night and daytime vision (see Oesch and Diamond, 2010). A manuscript is in preparation with the initial physiological work, and we have begun to collaborate with Dr. Kevin Briggman to mine densely reconstructed retinal tissue to identify changes in the relevant retinal circuitry. Finally, we are extending our electron microscopy studies, in collaboration with Tom Reese and Richard Leapman, to explore the detailed ultrastructure of synaptic ribbons in photoreceptors and rod bipolar cells. So far, EM tomography enables us to detect protein filaments that tether synaptic vesicles to the ribbon and the presynaptic membrane. This approach may enable us to discern morphologically, for the first time, docked and primed synaptic vesicles. We have applied a similar analysis to hair cell ribbon synapses in the auditory system and found postsynaptic specializations that optimize signal transfer at these synapses (manuscript submitted).